title: [Comparison of site-mutated and error-prone PCR methods for constructing the secondary phage antibody libraries].
abstract: OBJECTIVE
  To compare site-mutated PCR and error-prone PCR methods in constructing the secondary phage antibody libraries derived from a primary extracellular domain of cell adhesion molecule L1 (L1-ecd) binding single-chain variable fragment (scFv) antibody.
  
  
  METHODS
  Secondary mutant phage libraries were established by transfecting the mutated phage at the DNA level to E.coli TG1 with designed site-mutated PCR or error-prone PCR primers. Using the selected phagemid as the template, the mutated plasmid was amplified by PCR and then constructed with restriction enzyme cutting and ligation. Phage-based ELISA was used to calculate the ratios of the positive monoclones from the two libraries and the results were statistically compared using the Pearson x(2); method.
  
  
  RESULTS
  The size of the two libraries were 1.4×10(6); pfu/mL (site-mutated library) and 2.5×10(6); pfu/mL (error-prone library), respectively. The ratios of positive clones were 32.5% and 35.5%, respectively. The P value was 0.67, showing no significant difference.
  
  
  CONCLUSIONS
  These two methods can be widely used to obtain antibodies with a high affinity on the basis of the existing phage antibody.
authors.affiliation: Center for Neuroscience, Medical College, Shantou University, Shantou 515041, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Weijiang
authors.initials: W
authors.lastname: Zhao
mesh_terms: D004797:Enzyme-Linked Immunosorbent Assay; D039842:Neural Cell Adhesion Molecule L1 / Q000276:immunology*; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D057127:Single-Chain Antibodies / Q000235:genetics*
chemical_list: D039842:Neural Cell Adhesion Molecule L1; D019151:Peptide Library; D057127:Single-Chain Antibodies